PROJECT SUMMARY Transplant arteriosclerosis (TA) is a highly prevalent complication of heart transplantation and is the leading cause of late graft failure. TA lesions cause diffuse, irreversible narrowing of graft coronary arteries and account for an annualized rate of graft loss of ~5% per year. There are no effective medical therapies for TA, reflecting deficits in the understanding of its underlying causes. Donor specific antibody (DSA) binding MHC on graft endothelial cells (EC) is a strong risk factor for TA, but the mechanism(s) of how DSA causes TA are not well understood. To study this problem, DSA was modeled on EC using high panel reactive antibody (PRA) sera taken from sensitized transplant candidates. PRA sera deposited DSA on EC and activated complement, a system of immune-related proteins that when activated forms pore-like structures called membrane attack complexes (MAC) on cell surfaces. PRA caused MAC assembly on EC which induced inflammatory genes and increased the ability of EC to recruit and activate CD4+ T cells producing IFN-?, a vasculopathic cytokine. These processes had an overall effect of exacerbating TA lesions in human coronary arteries in a humanized mouse model. MAC mediated these effects through a novel effector pathway involving NF-?B-inducing kinase (NIK), a critical mediator of non-canonical NF-?B signaling. This proposal defines mechanisms of how MAC activates NIK in EC, explores how these mechanisms operate in a humanized mouse model for TA, and uses NIK in patient EC as a platform to develop novel diagnostic tests for TA. In Aim 1, endocytosis of MAC will be explored as a mechanism for NIK stabilization. MAC+ subcellular compartments will be isolated and characterized to identify novel non-canonical NF-?B signaling elements, Akt activation in MAC+ compartments will be explored as a mechanism for stabilizing NIK, and off-label use of FDA-approved drugs inhibiting endocytosis and Akt will be tested in vivo in a humanized mouse model of TA. Aim 2 explores clinical correlations between NIK and TA. The positive predictive value of NIK expression in two microvessel EC compartments will be assessed in patient samples, and novel markers correlating with TA will be identified using laser capture and RNA profiling of NIK+ EC in patient biopsy specimens. The long-term goal of this work is to improve outcomes for patients with TA. To this end the present application uses patient-oriented approaches to exploit novel mechanisms of TA to identify clinically relevant therapies and to develop diagnostic tests for this condition.